RNA Isolation - Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues

Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):

• D01 C

• D02 C

• D19 C

• D20 C

• T01 C

• T02 C

• T19 C

• T20 C

RNA was isolated using RNAzol RT (Molecular Research Center) in the following way:

• Unweighed, frozen tissue transferred to 500uL of RNAzol RT and immediately homogenized with disposable pestle.

• Added additional 500uL of RNAzol RT and vortexed to mix.

• Added 400uL of 0.1% DEPC-treated H2O, vortexed and incubated 15mins at RT.

• Centrifuged 12,000g for 15mins at RT.

• Transferred 750uL of supernatant to clean tube (discarded remainder), added 1 volume (750uL) of isopropanol, vortexed, and incubated at RT for 10mins.

• Centrifuged 12,000g for 10mins at RT.

• Discarded supernatant.

• Washed pellet with 75% ethanol (made with 0.1% DEPC-treated H2O).

• Centrifuged 4,000g for 2mins at RT.

• Discarded supernatant and repeated wash steps.

Pellet was resuspended in 50uL of 0.1% DEPC-treated H2O and stored @ -80oC in Ronit’s temporary box.